Clamping Procedure Research Articles

Method for manipulation of cytosolic pH in cells clamped in the whole cell or perforated-patch configurations

A number of methods have been developed to manipulate the intracellular pH (pHi) of intact cells. However, such methods are not applicable when cells are studied using the patch-clamp technique, due to the continuity of the cell interior with the recording pipette. The perfused-pipette method can be used to modify pHi in the whole cell configuration, but this approach is slow, technically demanding, and not useful in the case of the perforated-patch configuration. In this report, we introduce a simple procedure that enables the investigator to predictably and reversibly alter pHi in cells clamped in either the whole cell or perforated-patch modes. The method is based on the provision of a virtually unlimited reservoir of an intracellular H+ (equivalent) donor/acceptor system, by inclusion of large concentrations of permeable weak electrolytes in the pipette solution. This system not only provides a means for the imposition and maintenance of a chosen pHi but, by changing the external concentration of the weak electrolyte, enables the investigator to rapidly and reversibly change pHi or the transmembrane delta pH during the course of an experiment. The effectiveness of the procedure was validated in peritoneal macrophages by two methods: 1) direct measurement of pHi in single cells by fluorescence ratio determinations and 2) estimation of the reversal potential of H(+)-selective currents. The pHi clamping procedure is shown to be effective using either organic or inorganic weak bases in the whole cell configuration. In addition, because NH+4/NH3 can readily permeate the pores formed by nystatin or amphotericin, the method is also shown to apply to the perforated-patch configuration.

Cortical lesions attenuate the opposing effects of amphetamine and haloperidol on neostriatal neurons in freely moving rats.

Neuronal activity was recorded from the neostriatum of freely moving rats at least 1 week following either sham or bilateral ablations of frontal and somatosensory cortex. In both groups of animals, the majority of neurons increased firing rate in close temporal association with spontaneous movement. No group differences emerged either with respect to baseline firing rates or open-field behavior. Following amphetamine administration, however, the excitatory response of motor-related neurons was suppressed in cortical-lesioned rats. A behavioral clamping procedure, which assessed neuronal activity during matched pre- and post-amphetamine behaviors, confirmed these results, suggesting that the amphetamine-induced changes in neuronal activity reflect a direct drug effect independent of behavioral feedback. In animals that received a subsequent injection of 1.0 mg/kg haloperidol, cortical lesions attenuated the ability of this neuroleptic to block both the behavioral and neuronal effects of amphetamine. Collectively, these results support mounting evidence for an important modulatory influence of cortical afferents on the amphetamine-induced excitation of neostriatal neurons and the reversal of this effect by haloperidol.

Thermal Diffusion of Hydrogen and Hydride Precipitation in Zr–Nb Pressure Tube Alloys

AbstractThe thermal diffusion of hydrogen in a Zr–2.5 wt % Nb pressure tube alloy has been studied in the H range from 28 to 108 ppm as a function of specimen orientation. The heat of transport Q*, lay between 4.1 and 5.1 kcal/mol, and no effect of either H concentration or specimen orientation was detected in the range studied. A number of experimental runs (with the cold zone below the solubility limit for precipitation) were done to study hydride precipitation effects. The position at which coarse hydrides precipitated within the cold zone was determined by the thermal gradient. Stress reorientation was observed in circumferentially oriented specimens, associated with stresses induced by the specimen clamping procedure. A I.D. finite difference computer program, together with the concept of two solid solubilities (one for precipitation, the other for dissolution), was used to explain the experimental results.

The Onset and Progression of Pancreatic Insulitis in the Overt, Spontaneously Diabetic, Young Adult BB Rat Studied by Pancreatic Biopsy

A specially developed clamping procedure permitted the easy, complication-free removal of splenic pancreas from rats. Using this biopsy procedure pancreatic tissue was removed from 50- to 90-day-old BB rats to study in a retrospective experimental design the time at which insulitis appears in BB rats, which develop acute, overt diabetes before the age of 120 days. Islets in biopsies taken 18-53 days before the onset of diabetes showed normal structure and were free from any mononuclear infiltrations. Biopsies removed between 2 and 9 days before onset of diabetes in contrast showed widespread insulitis. In five rats in which the biopsy preceded the manifestation of diabetes by 11-16 days, only a small number of pancreatic islets showed small focal mononuclear cell infiltrations. Most of the islets in these five rats had a normal histologic appearance. Thus the lesions within the islets develop rapidly starting about 2-3 wk before overt diabetes. As revealed by autoradiography, pancreatic beta-cells still surviving at the time of onset of diabetes show a modest increase in replicative activity. Replicative activity of mononuclear inflammatory cells also was observed, suggesting that their accumulation within the islet tissue may result in part from local replication.

Change in the in vivo hyperthermic response resulting from the metabolic effects of temporary vascular occlusion

Previous workers have reported that clamping of animal tumors in vivo enhanced the effect of hyperthermia; the enhancement has been attributed to pH and nutritional effects of vascular occlusion. It has not been clear, however, the degree to which improved heating patterns or effects on the tumor cells and vasculature from the clamping procedure itself might have contributed to the observed effect. In the experiments herein reported, care was taken to insure comparable heating of C3H mouse mammary tumors transplanted on the flank whether clamped or unclamped. Clamping for one hour with hyperthermia during the final 30 minutes caused a marked thermosensitization as measured by tumor control. The temperature at 30 minutes heating to control 50% of the tumors for 120 days (TCT 50–120) was reduced from 46.8°C in controls to 43.5°C in clamped tumors, a difference of 3.3 ± 0.09°C. No cytotoxicity from the clamping alone was evident by assessment of subsequent tumor growth and no lasting vascular effects could be detected by 133Xe washout and tumor growth. Since the techniques used produced essentially identical heating patterns, we conclude that the striking enhancement in hyperthermic response in clamped tumors can be attributed to the metabolic consequences of temporary vascular occlusion.

Development of the Biostator Glucose clamping algorithm.

The "glucose clamping" technique has been proposed as a method for the early detection of a beginning derangement of glucose homeostasis and thus for the possible prevention of maturity-onset diabetes. This technique interrupts the physiological glucose-insulin relationship by placing a patient's blood glucose concentration under an investigator's control, for quantification of the pancreatic beta-cell response during hyperglycemic clamps and of sensitivity of body tissue to exogenous insulin during normoglycemic clamps. We report the development of a glucose clamping algorithm for use with the Biostator glucose-controlled insulin-infusion system (Horm. Metab. Res., Suppl. 8: 23-33, 1977). This algorithm adds simplicity and precision to the glucose clamping procedure and reduces operator effort to a minimum. We describe the early development of the algorithm with a model system and report evaluations made during animal studies and preliminary investigations with human subjects.

Modification of instrumentation for research on lingual vibrotactile sensitivity: elimination of the tongue clamping procedure.

A simplified modification in lingual vibrotactile instrumentation involving elimination of the tongue clamping procedure is presented. Reasons for this modification and the advantages are discussed in terms of facilitating clinical testing.

Neuronal-vascular relationship in experimental ischaemic anoxia.

The concept of temporal and spatial relationship between neuronal and vascular changes in the central nervous system following different kinds of anoxia still remains debatable, in that it is uncertain if anoxia-ischaemia primarily produces vascular changes in the brain or it initially damages the cerebral neurons. The present investigation has been undertaken to denote the sequential relationship between neuronal and vascular changes following experimental cerebral ischaemic anoxia. Adult healthy albino rats were subjected to ischaemic anoxia by bilateral clamping of their common carotid arteries for varying intervals of 5, 10 and 15 min. The animals of each group were subsequently sacrificed on days 1, 3, 5, 7 and 10 after the clamping procedure, for light-microscopic study of their brains. Our findings are characterized by initial vascular changes, which appear to have resulted in 'no-reflow' leading subsequently to neuronal damage. The latter, in turn, secondarily caused damage to the microvasculature.

The role of the circle of Willis in graded occlusion of the internal carotid artery in man.

Protracted studies on bilateral internal carotid artery (ICA) flow using implantable electromagnetic flow probes were performed in ten patients undergoing graded occlusion for infraclinoid saccular aneurysm. The ICA flow was generally lower on the side of the aneurysm in the eight patients with previous subarachnoid haemorrhage, and the difference was statistically significant at the 5% level. This was attributed to the increased vascular resistance in the affected hemisphere. A contralateral ICA flow increase, varying between 14 and 73%, was found in all patients at test occlusion of the ipselateral ICA. Three patients showed electro-encephalographic evidence of intolerance to clamping of the artery. They also showed the lowest contralateral compensatory increase (14–17%) at test occlusion. Furthermore, a delay in contralateral flow response to stepwise trial occlusion was observed in these patients. Two of these patients suffered ischaemic attacks in spite of special precautions taken during the subsequent graded clamping procedure. Complications also occurred in two patients who showed a satisfactory response to test oclusion. These complications, however, were found to be caused by thrombo-embolic processes in the ipselateral ICA during the period of graded occlusion and were not related to insufficient collaterals.

Selective inhibition of the transmembrane Ca conductivity of mammalian myocardial fibres by Ni, Co and Mn ions.

According to earlier studies on mammalian papillary muscles, Ni and Co ions reduce the Ca dependent mechanical response whilst the action potential persists. In order to clarify the mode of action of these cations together with those of Mn their effects on the transmembrane fast Na and of the slow Ca currents were studied in voltage clamp experiments using the double sucrose gap technique. By a conditioning clamping procedure the Ca current was separated from the Na current. The following results were obtained: 1. Ni ions (2 mM) and Co ions (2 mM) strongly depress the Ca conductivity of the membrane. The same blockade was observed under the influence of Mn ions (2 mM). Even in the continued presence of Ni, Co or Mn the transmembrane Ca conductivity can be restored to a varying degree by increasing the extracellular Ca concentration. 2. In contrast to this strong inhibitory effect on the transmembrane Ca inward movements the fast transient Na current is only slightly affected if at all. 3. Apart from their selective inhibitory action on the transmembrane Ca current, Co ions show some additional features a) enhancement of its inhibitory action by repeated membrane depolarizations. b) Co-induced replacement of the slow Ca inward current by a transitory fast inward current component, c) synergetic effects of Co with Ca in restricting the fast Na current. 4. The selective inhibition of the transmembrane Ca conductivity clearly demonstrates the existence of a separate channel for Ca which is independent from the fast Na channel of the membrane.

Modification of Irradiation Effects on Rat Embryos by Uterine Vascular Clamping

Uterine vascular clamping of the pregnant rat uterus for 45 minutes protects against the lethal and growth-retarding effects of irradiation on the eighth day of gestation and against the lethal, growth-retarding, and probably teratogenic effects of irradiation on the ninth day of gestation. If the clamping procedure is continued for more than an hour on the eighth and ninth days; it results in a high incidence of malformations. The most likely basis for the protective effect of the clamping procedure is the resultant hypoxia, but the definitive answer is not yet available. The inability of irradiation to induce malformations in the 8-day 0-hour-old rat embryo was confirmed. The discrepancy between the teratogenic action of vascular clamping and irradiation on the eighth day is presented and discussed. (auth)

Induction of Intestinal Carcinoma in the Rat by X-Irradiation of the Small Intestine

The exteriorized ileum and jejunum of maie Holtzman rats were x irradiated with doses from 1000 to 2500 r. The superior mesenteric artery and vein were clamped during radiation exposure in about haif of the irradiated animals. Thirty-day survival and appearance of intestinal tumors in the 12 months post-irradiation were noted. Per cent survival at 30 days was increased by the blood restriction procedure in all cases where there was some mortality due to irradiation alone. Over 50% of the irradiated animals that survived exposure doses above 1400 r because of the clamping procedure developed intestinal tumors. Radiation alone induced the neoplasm in only the 1400-r group. Tumors appeared as early as 55 days and as late as 225 days postirradiation. Animals without surgery or irradiation and those subjected to the intestinal vessel clamping procedure alone did not develop tumors. The neoplasm has features of adenocarcinoma including invasion of submucosal and muscle layers of the gut, but the possibility that the apparent invasion of fibrous tissue by epithelium is due to trapping of epithelium still exists in some animals. Glandular epithelium in the diaphragm of one rat with an intestinal tumor and apparent neoplastic glands in the gut mesentery ofmore » another was found. There is little, if any, doubt that these represent metastases. It was not possible to transplant the tumor into normal animals. (auth)« less

THE EFFECT OF PREVIOUS EXPOSURE TO COLD ON SHOCK SECONDARY TO LIMB ISCHAEMIA

Previous exposure to a cold environment for a period of 12 weeks increased the incidence of survival and prolonged the survival time of heavy rats shocked by a clamping technique. The oxygen consumption at 27 °C in rats previously exposed to cold for 12 weeks was elevated. During the clamping period the oxygen consumption became more nearly the same in the cold-acclimatized and control rats. After release of the clamps there was a slower fall in oxygen consumption in the previously cold-acclimatized rats than in the control animals. The body temperature, measured in the colon, fell more slowly in the previously cold-exposed rats than in the control rats. This slower fall in colonic temperature was found also in rats previously exposed to cold for shorter periods and was not necessarily associated with changes in survival time. Rats exposed to cold for 6 weeks were "acclimatized" in the sense that they withstood exposure to cold after removal of their fur, but they failed to show significantly lengthened survival times when shocked by this clamping procedure. The administration of thyroxine shortened survival times in the shocked rats but reduced the rate of fall of colonic temperature. The administration of cortisone, thyroxine, and cortisone plus thyroxine shortened survival times in the shocked rats.

THE EFFECT OF PREVIOUS EXPOSURE TO COLD ON SHOCK SECONDARY TO LIMB ISCHAEMIA

Previous exposure to a cold environment for a period of 12 weeks increased the incidence of survival and prolonged the survival time of heavy rats shocked by a clamping technique. The oxygen consumption at 27 °C in rats previously exposed to cold for 12 weeks was elevated. During the clamping period the oxygen consumption became more nearly the same in the cold-acclimatized and control rats. After release of the clamps there was a slower fall in oxygen consumption in the previously cold-acclimatized rats than in the control animals. The body temperature, measured in the colon, fell more slowly in the previously cold-exposed rats than in the control rats. This slower fall in colonic temperature was found also in rats previously exposed to cold for shorter periods and was not necessarily associated with changes in survival time. Rats exposed to cold for 6 weeks were "acclimatized" in the sense that they withstood exposure to cold after removal of their fur, but they failed to show significantly lengthened survival times when shocked by this clamping procedure. The administration of thyroxine shortened survival times in the shocked rats but reduced the rate of fall of colonic temperature. The administration of cortisone, thyroxine, and cortisone plus thyroxine shortened survival times in the shocked rats.

THE METABOLISM OF THE KIDNEY IN EXPERIMENTAL RENAL HYPERTENSION

These investigations are part of an attempt to study and interpret the intermediary metabolism of the kidneys in experimental renal hypertension. Hypertension was produced in dogs by the clamping procedure of Goldblatt and associates or by the silk perinephritis method of Page. Enzymatic studies were made by means of Warburg's manometric method. Cytochrome c was in addition determined spectrophotometrically. Tissue slices, homogenized tissue, and tissue extracts were used. A study of the cytochrome c concentration and the activities of the cytochrome oxidase and succinic dehydrogenase systems of kidneys from normal dogs and dogs with experimental renal hypertension was made. It was found that the cytochrome c concentration and the activities of the cytochrome oxidase and succinic dehydrogenase systems were markedly lower in the kidney slices and in the tissue suspensions from hypertensive dogs. Tissue suspensions and extracts of kidneys from hypertensive dogs showed an inhibitory effect on the activity of the cytochrome oxidase and succinic dehydrogenase, and the amine oxidase systems. Renin preparations also showed a marked inhibitory effect on the activities of cytochrome oxidase, succinic dehydrogenase, l-amino acid oxidase, and amine oxidase systems. A significant increase was found in the kidney of dogs whose other kidney had been removed or subjected to Goldblatt's or Page's technique in the activities of the cytochrome-cytochrome oxidase system, the succinic dehydrogenase system, and in the concentration of nucleotide-bound phosphorus, of flavin-adenine dinucleotide, and of the nicotinamide-adenine dinucleotides (coenzymes I and II). From the results of these studies it can be concluded that an increase in the concentration and activity of the respiratory enzymes precedes hypertrophy of the kidney. This can be explained by the assumption that an increase in the activity of the respiratory biocatalysts acts as a stimulus for cell growth and multiplication.